1. Field of the Invention
The present invention is directed to novel compounds useful as inhibitors of heat shock 90 proteins (“Hsp90”), the molecular chaperones responsible for protein folding and maturation in vivo and which have been found at higher levels in cancerous cells than in normal cells. The compounds of the present invention have a chimeric structure based, in part, on two naturally occurring Hsp90 inhibitors: radicicol (“RDC”) and geldanamycin (“GDA”).
2. Description of Related Art
The 90 kDa heat shock proteins belong to a family of chaperones that regulate intracellular functions and are required for the refolding of denatured proteins following heat shock, as well as the conformational maturation of a large number of key proteins involved in cellular processes. The Hsp90 family of chaperones is comprised of four different isoforms. Hsp90 α and Hsp90 β are found predominately in the cytosol, the 94-kDa glucose-regulated protein (“GRP94”) is localized to the endoplasmic reticulum, and Hsp75/tumour necrosis factor receptor associated protein 1 (“TRAP-1”) resides mainly in the mitochondrial matrix. These Hsp90s bind to client proteins in the presence of cochaperones, immunophilins, and partner proteins to make the multiprotein complex responsible for conformational maturation of newly formed nascent peptides into biologically active three-dimensional structures.
As discussed more fully below, Hsp90 is an ATP-dependent protein with an ATP binding site in the N-terminal region of the active homodimer. Disruption of the ATPase activity of Hsp90 results in the destabilization of multiprotein complexes and subsequent ubiquitination of the client protein, which undergoes proteasome-mediated hydrolysis. More specifically, in an ATP-dependent fashion, Hsp70 binds to newly synthesized proteins cotranslationally and/or posttranslationally to stabilize the nascent peptide by preventing aggregation. Stabilization of the Hsp70/polypeptide binary complex is dependent upon the binding of Hsp70 interacting protein (“HIP”), which occurs after Hsp70 binds to the newly formed peptide. Hsp70-Hsp90 organizing protein (“HOP”) contains highly conserved tetratricopeptide repeats (“TPRs”) that are recognized by both Hsp70 and Hsp90, promoting the union of Hsp70/HIP and Hsp90, which results in a heteroprotein complex. In the case of telomerase and steroid hormone receptors, the client protein is transferred from the Hsp70 system to the Hsp90 homodimer with concomitant release of Hsp70, HIP, and HOP. Upon binding of ATP and an immunophilin with cis/trans peptidyl prolyl-isomerase activity (FKBP51, FKBP52, or CyPA), the ensemble folds the client protein into its three-dimensional structure. In a subsequent event, p23 binds Hsp90 near the N-terminal region promoting the hydrolysis of ATP and release of the folded protein, Hsp90 partner proteins, and ADP.
Examples of proteins dependent upon Hsp90 for conformational maturation include oncogenic Src kinase, Raf, p185, mutant p53 (not normal p53), telomerase, steroid hormone receptors, polo-like kinase (“PLK”), protein kinase B (“AKT”), death domain kinase (“RIP”), MET kinase, focal adhesion kinase (“FAK”), aryl hydrocarbon receptor, RNA-dependent protein kinase (“PKR”), nitric oxide synthase (“NOS”), centrosomal proteins, and others. In addition, other proteins, such as cyclin dependent kinase 4 (“CDK4”), cyclin dependent kinase 6 (“CDK6”), and human epidermal growth factor receptor 2 (“Her-2”) are thought to be client proteins of Hsp90. Of these Hsp90 client proteins, Raf, PLK, RIP, AKT, FAK, telomerase, and MET kinase are directly associated with the six hallmarks of cancer: (1) self-sufficiency in growth signals; (2) insensitivity to antigrowth signals; (3) evasion of apoptosis; (4) unlimited replication potential; (5) sustained angiogenesis; and (6) tissue invasion/metastasis. Consequently, Hsp90 is a target for the development of cancer therapeutics because multiple signaling pathways can be simultaneously inhibited by disruption of the Hsp90 protein folding machinery.
Known inhibitors of Hsp90 include the anti-tumor antibiotics geldanamycin (“GDA”), radicicol (“RDC”), herbimycin A (“HB”), a 17-allylamino derivative of GDA (“17-AAG”), and the synthetic ATP analog called PU3. The structures of these prior art Hsp90 inhibitors are shown in FIG. 1.
These prior art molecules exert their activity by binding to the N-terminal ATP binding pocket and inhibit the ATPase activity of Hsp90. The energy normally derived from ATP hydrolysis is used to elicit a conformational change that releases the properly folded client protein from Hsp90. However, when a non-hydrolyzable inhibitor is present, Hsp90 is unable to fold the bound client protein, resulting in ubiquitination of the client protein and subsequent proteolysis by the proteasome.
As shown in FIG. 2, when the co-crystal structures of GDA and RDC were solved, it was determined the quinone ring of GDA and the 2,4-diphenol of RDC bound in opposite orientations. See Roe et al., Structural Basis for Inhibition of the Hsp90 Molecular Chaperone by the Antitumor Antibiotics Radicicol and Geldanamycin, J. Med. Chem. 1999, 42, 260–266, which is incorporated by reference in its entirety. The 2,4-dihydroxy moiety of RDC binds in the same location as the adenine ring of ADP and mimics the hydrogen bond donor/acceptor properties of the exo- and N7 endocyclic amines. The quinone ring of GDA binds towards the exterior of the pocket and facilitates hydrogen bond interactions with the diphosphate-binding region. The Kd of GDA and RDC is estimated to be about 1200 and 19 nM, respectively.
Several Hsp90 inhibitors have been investigated therapeutically. GDA has potent activity in vitro with an IC50 of 1–3 μm. However, in vivo GDA has a greater affinity for the Hsp90 complex with an IC50 of 100 nM. As a result, a derivative of GDA, 17-AAG, has entered Phase I clinical trials for the treatment of several cancers.
Several modifications to the GDA and RDC structures have also been investigated therapeutically to some extent. For example, based on bovine Hsp90, researchers have suggested that removal of the C28 methyl substituent of GDA or incorporation of an H-bond donor into this position would lead to increased affinity. See Stebbins et al., Crystal Structure of Hsp90-Geldanamycin Complex: Targeting of a Protein Chaperone by an Antitumor Agent, Cell, 1997, 89, 239–250, which is incorporated by reference in its entirety. Modifications of the quinone moiety of GDA have demonstrated that replacement of the C17 methyl ether with amino side chains enhanced the biological activity as evidenced by lower IC50s via stabilization of the quinone, ring and the conservation of a hydrogen bond acceptor at this position. See Schnur et al., Inhibition of the Oncogene Product p185erb-2 in Vitro and in Vivo by Geldanamycin and Dihydrogeldanamycin Derivatives, J. Med. Chem. 1995, 38, 3806–3812; Schnur et al., erB-2 Oncogene Inhibition by Geldanamycin Derivatives: Synthesis, Mechanism of Action, and Structure-Activity Relationships, J. Med. Chem. 1995, 38, 3813–3820; Sasaki et al., Growth Inhibition of Virus Transformed Cells In Vitro and Antitumor Activity In Vivo of Geldanamycin and Its Derivatives, J. Antibiotics 1979, 32, 849–854, which are all incorporated by reference in their entirety. In addition, when the co-crystal structure of Hsp90 bound to GDA was solved, the authors suggested the meta-carbonyl was acting as a hydrogen bond acceptor. See Roe et al., Structural Basis for Inhibition of the Hsp90 Molecular Chaperone by the Antitumor Antibiotics Radicicol and Geldanamycin, J. Med. Chem. 1999, 42, 260–266, which is incorporated by reference in its entirety. Also, Santi and coworkers have recently reported the co-crystal structure of 17-dimethylaminoethylamino-geldanamycin (DMAG) bound to Hsp90 and performed a series of computational studies to understand the relationship between the bent conformation of GDA found in the Hsp90 binding site and its native conformation. See Jez et al., Crystal structure and molecular modeling of 17-DMAG in complex with human Hsp90, Chem. Biol. 2003, 10, 361–368, which is incorporated by reference in its entirety. Their studies suggest that GDA binds to Hsp90, and is twisted into a bent conformation by isomerization of the amide bond (trans→cis), which results in an enthalpic penalty between 2.2 and 6.4 kcal/mol. They further suggested that analogues of GDA that contain a cis-amide will have >1000 fold increase in affinity for Hsp90.
Although GDA and RDC have been shown to inhibit Hsp90 and some GDA and RDC derivatives have been proposed, there remains a need to develop other Hsp90 inhibitors as useful anti-cancer agents. Most preferably, these new Hsp90 inhibitors have decreased toxicity, increased solubility, and/or increased selectivity for Hsp90.
The present invention is directed to novel compounds having a chimeric structure comprised of moieties that mimic the binding regions of the prior art Hsp90 inhibitors. Most preferably, the novel compounds contain moieties that mimic the interactions of both GDA and RDC with Hsp90. These novel Hsp90 inhibitors bind to the N-terminal ATP binding region of Hsp90, and have a predicted Kd in the submicromolar range (even in the nanomolar and picomolar range). Furthermore, the present invention also includes modified derivatives and analogues of the chimeric compounds. These derivatives include the seco-ester and seco-amide variations of the chimeric structure.
Additional aspects of the invention, together with the advantages and novel features appurtenant thereto, will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned from the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.